Small chemical moieties, such as heavy metal ions, can and often do affect the environment and biological systems. These effects become astounding when it is realized that minute quantities of these small moieties are involved. Moreover, the presence or absence of low concentrations of small moieties in the environment can have long term consequences. Minute quantities of metallic cations, such as lead cations, can regulate, influence, change or toxify the environment or biological systems.
The detection, removal, addition or neutralization of such minute quantities constitutes a focal point for continued research in many fields. For example, many efforts have been made to detect and remove minute, toxic amounts of heavy metal ions such as lead or mercury from the environment. The efforts often have not been successful or economical for widespread application. On the other hand, minute concentrations of other heavy metals are important for the proper function of biological organisms. Zinc, for example, plays a major role in wound healing.
Heavy metal can exhibit dual roles. For example, lead is used in glass making and in chemical manufacturing operations. Yet when ingested by mammals, such as from drinking water, lead may be highly toxic in very small amounts. Hence, detection and quantification of minute concentrations of a heavy metal, such as lead, in drinking water and other media would serve exploratory, safety and regulatory goals.
It would, therefore, be highly desirable to identify and control minute quantities of heavy metals, e.g., lead cations, in aqueous biological or inanimate systems. In most contexts, however, the detection, removal, addition or neutralization of heavy metals, is a difficult and expensive and often unfeasible if not impossible task. Other metallic contaminants often mimic the heavy metal of interest and result in measurement interference. Moreover, the detection methods employed today are usually not sufficiently sensitive at the minute quantities under consideration. Consequently, it is desirable to develop reliable and economic methods for accurately identifying and controlling minute quantities of a particular heavy metal in the presence of other heavy metals.
Antibodies would seem to be uniquely suited for this task. Their high degree of specificity for a known antigen would avoid the potential interference caused by contaminants. The sensitivity of antibodies in assays to detect analytes in the picomolar or lower range would permit accurate and efficient targeting and detection at minute levels.
Monoclonal antibodies, of course, come to mind as especially suited agents for practice of this technique. Since Kohler and Milstein published their article on the use of somatic cell hybridization to produce monoclonal antibodies (Nature 256:495 (1974)), immunologists have developed many monoclonal antibodies which strongly and specifically immunoreact with antigens.
Notwithstanding this suggestion, the conventional understanding about immunology teaches that antibodies against small moieties, such as heavy metals, cannot be developed. The mammal immunization step, which is key for the production of monoclonal antibodies, typically requires a molecule that is large enough to cause antigenic reaction. Medium sized molecules (haptens), which are not of themselves immunogenic, can induce immune reaction by binding to an immunogenic carrier. Nevertheless, immunologists view small moieties such as metallic cations, as not large or structurally complex enough to elicit an antibody response. The molecular size and lack of complexity of an inorganic cation is thought to render it insufficient for eliciting an antibody response.
Several immunologists have reported production of monoclonal antibodies to metallic ion chelates. For example, in U.S. Pat. No. 4,722,892, monoclonal antibodies are disclosed which immunoreact with a complex of a chelating agent, such as ethylene diamine tetracetate (EDTA), and a heavy metal such as indium. In EPO Patent Application 0235457, monoclonal antibodies that immunoreact with cyanide complexes of gold and/or silver are disclosed. In these instances, however, the monoclonal antibodies bind with the metal chelate complex rather than the bare metallic ion itself. Disadvantages of antigen measurement methods based on such antibodies include: the complicated reagents involved in detection, lack of simple tests that discriminate among antigens, cross-reactivity with chelates of other antigens and cross-reactivity with the chelating agent itself.
Other instances of monoclonal antibody combinations with metals involve metal tags. The metals or metal chelates are bound to the antibody at a site remote from the antigen binding site or sites. The metal tag is not the antigen. Instead, the metal tag is used to indicate the presence of the monoclonal antibody when it reacts with its specific antigen. See for example, V.P. Torchilian et al., Hybridoma, 6, 229 (1987); and C.F. Meares, Nuclear Medical Biology, 13, 311-318 (1986).
Accordingly, there exists a need to develop polypeptides that immunoreact with heavy metals per se and with lead ions in particular. This would permit the development of methods for detecting or neutralizing heavy metals within, adding heavy metals to, or removing heavy metals from biological or inanimate systems through the use of the monoclonal antibodies. There also exists a need for the development of nucleic acid sequences coding for polypeptides which selectively bind with lead cations and the development of methods of expressing and translating these nucleic acid sequences to produce metal binding polypeptides.